Small, free-living amoebae (FLA) are ubiquitous in nature and are found in soil, fresh water, and marine environments. Most of the FLA feed on bacteria and are of no medical importance, yet several genera and species of FLA are known to causes serious, usually fatal disease in humans and animals. Naegleria fowleri and Acanthamoeba spp. are the best known examples of pathogenic FLA. N. fowleri thrives in warm, freshwater and is the causative agent of primary amoebic meningoencephalitis (PAM). This disease is characterized by a fulminant, rapidly fatal encephalitic disease that most often afflicts healthy young humans. Acanthamoeba spp. are more ubiquitous and found in water (fresh and saline) and soil. Multiple species of Acanthamoeba are known to cause granulomatous amoebic encepahalitis (GAE), a chronic disease seen most often in immunocompromised hosts and those at risk of opportunistic infections. Acanthamoeba spp. also causes amoebic keratitis, skin, nasopharyngeal, and disseminated infections. The major problem for infections with any of the pathogenic FLA is the lack of effective therapeutics. PAM and GAE are usually fatal diseases, even if the infection is diagnosed promptly and treated with the best available drug regimens. Despite the severity of infections caused by FLA, there are few data available on new drugs and no concerted modern drug discovery or development efforts. The majority of the research literature on drug discovery for FLA consists of limited in vitro or in vivo studies with drugs already approved for other uses. The major goals of this project are to discover and develop and to late lead ? one new drug to treat central nervous system infections with pathogenic FLA. We have conducted structure activity studies with a series of amidino related compounds (>160) and identified two chemotypes for further development. In the R21 phase, we propose to conduct additional lead optimization to develop early lead candidates by the end of the R21 phase. Candidates will be prioritized based upon in vitro potency, physiochemical properties, and rate-of-killing in a novel dual cell viability assay.In the R33 phase we will further optimize for potency in vivo and enhancement of PK and ADME properties and conduct safety toxicology studies with the best late lead candidates. Our ultimate goal for this project is to identify one novel amidino related compound that can be used in combination with other drugs for the treatment of pathogenic FLA infections of the central nervous system.